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Abstract:

The present invention provides means and methods for simple and efficient
introduction of foreign genetic material into the plant cell.
Particularly, the present invention combines seed priming and virus-based
DNA constructs for efficient introduction of heterologous DNA into
plants.

Claims:

1. A method for introducing heterologous DNA into at least one cell of a
plant seed embryo comprising contacting a plant seed with a priming
medium containing a virus-based DNA construct comprising the heterologous
DNA under conditions enabling priming, said plant seed comprising a seed
coat, thereby obtaining the seed embryo comprising the heterologous DNA.

2. The method of claim 1, further comprising the step of providing
suitable conditions for subsequent seed germination and growth so as to
obtain a plant or part thereof comprising the heterologous DNA.

12. The method of claim 11, wherein the DNA construct is a
Geminivirus-based construct selected from the group consisting of IL-60
having the nucleic acids sequence set forth on SEQ OD NO:1 and IL-60-BS
having the nucleic acids sequence set forth in SEQ ID NO:2.

13. The method of claim 1, wherein the DNA construct is an expression
construct thereby the heterologous DNA is expressed within the plant
cell.

14. (canceled)

15. (canceled)

16. (canceled)

17. The method of claim 16, wherein the heterologous DNA encodes a
product useful in the cosmetic or pharmaceutical industry.

18. (canceled)

19. The method of claim 1, wherein the heterologous DNA encodes a product
which confers a desirable agronomic trait selected from the group
consisting of resistance to biotic or abiotic stress, increased yield,
increased yield quality and preferred growth pattern.

20. The method of claim 1, wherein the heterologous DNA is transiently
expressed in the at least one cell of the seed embryo and cells derived
therefrom.

21. The method of claim 1, wherein the heterologous DNA is incorporated
into the genome of the at least one cell of the seed embryo and cells
derived therefrom.

22. The method of claim 1, wherein the priming medium have a water
potential enabling seed imbibitions but not radicle emergence.

23. The method of claim 22, wherein the priming medium is an aqueous
solution.

24. The method of claim 22, wherein the priming medium is a solid matrix.

25. A seed which comprises a virus-based DNA construct comprising
heterologous DNA, wherein said heterologous DNA was introduced into the
seed by contacting a plant seed which comprises a seed coat with a
priming medium containing said virus-based DNA construct comprising said
heterologous DNA under conditions enabling priming.

26. A plant or part thereof grown from the seed according to claim 25,
wherein the plant or part thereof comprises the virus-based DNA construct
comprising heterologous DNA.

27. The method of claim 1, wherein the seed is of a monocot origin.

28. The method of claim 1, wherein the seed is of a dicot origin.

29. The method of claim 1, wherein the seed is of cone-bearing origin.

Description:

FIELD OF THE INVENTION

[0001] The present invention relates to the field of plant molecular
biology, particularly to means and methods for simple and efficient
introduction of exogenous genetic material into the plant cell.

BACKGROUND OF THE INVENTION

[0002] Plants carrying one or more expressible heterologous genes have a
variety of potential advantages. The plants carrying a gene expression
cassette may carry one or more genes which confer desired traits,
including for example, herbicide, pesticide or insect tolerance;
tolerance to stress; enhanced flavor and/or shelf life of the fresh
produce (fruit, vegetable, seeds etc.), as well as the ability to amplify
the synthesis of useful plant endogenous and/or foreign proteins, sugars,
fatty-acids or secondary metabolites for consumption by man and/or
animals or for use as raw materials in a variety of industries
(cosmetics, pharmaceuticals, nutraceutics, foods, paper, fibers, etc.).

[0003] Current transformation technologies provide an opportunity to
engineer plants with desired traits, and major advances in plant
transformation have been occurred in recent years. The most common
technology is mediated by Agrobacterium tumefaciens. Other methods of
transformation are direct DNA transfer including microinjection,
electroporation, particles bombardment and viral vectors (Birch R G.
1997. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:297-326). With the
exception of viral vectors, application of the above techniques results
in the penetration of foreign DNA into the treated cells/tissue, its
integration into the treated plant's genome and the regeneration of
transgenic plants. However, in many major crop plants, serious genotype
limitations still exist. Transformation of some agriculturally important
crop plants continues to be both difficult and time consuming. In
addition, in all the aforementioned technologies, transformation is
conducted with vegetative tissues (including embryos) the efficiency of
transformation is poor, markers for selection are required, and the
percentage of successful regeneration of a plant from the transformed
cell or tissue is rather low.

[0004] Integration of the foreign DNA into the plant genome is not always
desirable, particularly when genetically modified (GMO) plants arouse
environmental and political issues. In these cases, methods enabling the
expression of the heterologous gene(s) that are not incorporated into the
host genome are required.

[0005] International (PCT) Application Publication No. WO 2007/141790 to
some of the inventors of the present invention discloses modified
Geminivirus-based constructs capable of spreading from one plant cell to
the other within the treated plant and concomitantly introducing foreign
DNA into the plant cells, without exerting the pathological virus
symptoms. This foreign DNA is expressed in the plant tissues but is not
integrated into its genome. The constructs (also referred to as
expression vectors, IL-60 being one example) comprise the heterologous
polynucleotide sequence that interrupts the Geminivirus replicase genes
such that it is flanked by a non-contiguous nucleic acid sequence
encoding a Geminivirus replicase or replicase-associated protein.

[0006] Seed priming is a process for treating plant seeds that enables
them to undergo faster and more uniform germination on sowing or planting
compared to non-treated seeds. Additionally, priming offers an optional
simultaneous treatment with fungicide/pesticides/fertilizers or other
chemicals (e.g. coating, pelleting, coloring as a trade mark, and more)
providing protection and/or facilitating germination, emergence and
seedlings establishment.

[0007] Priming allows the seeds to absorb enough water to enable their
pre-germinative metabolic processes to begin and then arrests them at
that stage. The amount of water absorbed (with or without beneficial
chemicals) must be carefully controlled, as too much would simply allow
the seed to germinate and too little would result in the seed ageing.
Once the correct amount of water has been absorbed, the seeds may be sown
or dried back to the original water content for storage. The primed seeds
usually germinate and emerge more quickly and uniformly and the seedling
vigor is typically higher compared to unprimed seeds, particularly under
suboptimal conditions. The benefits gained by priming, such as rate and
uniformity of germination are usually attributed to the initiation of
DNA-repair processes, protein hydration enzyme activation and additional
processes occurring in the early stages of germination.

[0008] The three major techniques used for controlled water uptake include
priming with aqueous solutions, with hydrated solid particulate systems
or by controlled hydration with water. Priming with solution is based on
immersing the seeds in osmotic solution, typically PEG solutions
characterized by osmotic potential that enables limited imbibition that
is insufficient for full hydration and seed germination. Alternatively,
the same effect is achieved by mixing the seeds with hydrated absorbent
medium such as clay or peat (e.g., U.S. Pat. No. 4,912,874). Controlled
hydration with aqueous solutions may be achieved, for example, by
utilizing semi-permeable membrane to mediate the transfer of water from a
solution of a given osmotic pressure to the seed (U.S. Pat. No.
5,992,091). Priming is performed under a variety of temperatures and
aeration methods (e.g., stirring, agitation, bubbling, etc.) using any of
the techniques for controlled water uptake (Taylor A G. et al. 1998. Seed
Science Technology 8:245-256).

[0009] Combination between the common transformation techniques described
hereinabove and seed priming has been disclosed. For example, U.S. Pat.
No. 6,646,181 discloses a method of introducing genes into plants
comprising synchronizing the stage of development of the plant at a stage
that includes large amounts of 4C DNA in seeds of the plants and
transfecting the cells of the seeds, in which synchronizing the stage of
development comprises admixing a particulate solid matrix material and a
seed priming amount of water, with aeration of seeds, for a time and at a
temperature sufficient to cause a substantial number of the cells of the
seeds to reach a desired stage of a cell cycle.

[0010] U.S. Patent Application Publication No. 2006/0005273 discloses
maize explants suitable for transformation. The explants comprise a maize
seed split in half longitudinally, wherein the splitting exposes the
scutellum, the coleoptilar ring and shoot apical meristem, each of which
are independently suitable for transformation. Priming the seed prior to
splitting with either callus or shoot priming medium increases the callus
and shoot induction frequency after transformation.

[0011] U.S. Patent Application No. 20100154083 discloses compositions and
methods to screen, identify, select, isolate, and/or regenerate targeted
integration events using seed priming. Seed priming provides the
identification of a seed having stably incorporated into its genome a
site-specific recombinase mediated integration of a selectable marker at
a target locus operably linked to a promoter active in the seed.

[0012] These combination methods employ the known methods of
transformation and are aimed at increasing the efficacy of foreign gene
integration into the plant genome. As described hereinabove, the
available methods have limitations with regard to applicability and
efficacy. In addition integration of the foreign DNA to the plant genome
is not always desirable.

[0013] There is a recognized need for, and it would be highly advantageous
to have means and methods for simple and efficient introduction of DNA
into plant cells that further enable efficient plant regeneration.

SUMMARY OF THE INVENTION

[0014] The present invention provides means and methods for simple and
efficient delivery of heterologous DNA into a plant cell, part, tissue,
organ, or the entire organism, particularly to the cells of a seed embryo
within an intact seed. The seed comprising the introduced heterologous
DNA is easily grown to a mature plant under standard growth conditions,
without the need of regeneration cultures and hardening conditions.

[0015] The present invention is based in part on the unexpected discovery
that introduction of heterologous DNA into plant seeds is possible during
seed priming by supplementing the priming medium with a virus-based DNA
construct, particularly with a Geminivirus based expression construct. As
exemplified hereinbelow, the present invention now shows that
supplementing a priming medium with a Geminivirus based construct
designated IL-60, which comprises the heterologous polynucleotide
sequence flanked by non-contiguous nucleic acid sequences encoding a
Geminivirus replicase or replicase associated proteins, result in the
uptake, replication and symptomless spreading of the DNA construct within
the seed cells.

[0016] The teachings of the present invention are advantageous over
previously known methods for plant cell transformation or introduction of
foreign DNA in that the DNA is introduced into the embryo of the intact
seed which is naturally capable of developing into a whole plant. The
teachings of the present invention for DNA introduction does not involve
damage to the cells (as occurs when direct DNA transfer, particularly via
bombardment is employed) or cell functioning. Furthermore, when
Geminivirus-based expression constructs are employed, the heterologous
DNA is not incorporated into the plant genome, again preventing the
trauma accompanying DNA introduction into the cell which typically leads
to cell cycle arrest and apoptosis or programmed cell death. The simple
methods of the present invention can be employed with seeds of any plant
of interest, and is fast and highly efficient. When the DNA construct
used is such that the heterologous DNA does not incorporate into the
plant genome, the method is further advantageous as the plant expresses
the desired product but is not defined as genetically modified.

[0017] Thus, according to one aspect, the present invention provides a
method for introducing heterologous DNA into at least one cell of a plant
seed embryo comprising contacting a plant seed with a priming medium
containing a virus-based DNA construct comprising the heterologous DNA
under conditions enabling priming, thereby obtaining the seed embryo
comprising the heterologous DNA.

[0018] According to certain embodiments, the method further comprises the
step of providing suitable conditions for subsequent seed germination and
growth so as to obtain a plant or part thereof comprising the
heterologous DNA.

[0020] According to typical embodiments, the DNA construct is a
Geminivirus based construct. According to these embodiments, the
construct comprises the heterologous DNA flanked by a non-contiguous
nucleic acid sequences encoding Geminivirus replicase or
replicase-associated protein.

[0021] According to other embodiments, the construct further comprises a
polynucleotide sequence encoding a modified Geminivirus coat protein
(CP). According to typical embodiments, the modified Geminivirus coat
protein encoding polynucleotide comprises a mutation or deletion in
nucleotides encoding the N-terminal 100 amino acids.

[0022] According to further typical embodiments the expression construct
further comprises a polynucleotide sequence encoding a modified
Geminivirus V2 protein.

[0023] According to additional typical embodiments the expression
construct further comprises a polynucleotide sequence encoding a modified
Geminivirus C4 protein. According to these embodiments, the modified
Geminivirus C4 protein includes a mutation or deletion. According to
certain embodiments the expression construct further comprises a
bacterial polynucleotide sequence.

[0024] According to certain currently preferred embodiments, the
Geminivirus is Tomato Yellow Leaf Curl Virus (TYLCV). According to these
embodiments, the Geminivirus-based construct is selected from the group
consisting of IL-60 having the nucleic acids sequence set forth on SEQ OD
NO:1 and IL-60-BS having the nucleic acids sequence set forth in SEQ ID
NO:2.

[0025] According to certain embodiments, the DNA construct is designed as
an expression construct such that the heterologous DNA is expressed in
the plant cell. According to these embodiments, the DNA construct further
comprises at least one regulatory element selected from the group
consisting of an enhancer, a promoter, and a transcription termination
sequence.

[0026] According to additional embodiments, the DNA construct further
comprises a marker for identifying the seeds and/or plants comprising the
heterologous DNA.

[0027] The heterologous DNA can encode any desired product, including
peptides, polypeptide, proteins and RNAs. The proteins or RNAs can be of
plant origin or of other origin, including but not limited to bacterial
and mammal origin. According to certain embodiments, the DNA encodes an
inhibitory RNA selected from the group consisting of antisense RNA,
dsRNA, siRNA and the like, such that the expression of a target gene is
silenced. According to other embodiments, the heterologous DNA encodes a
product the expression of which confers a desirable agronomic trait
including, but not limited to, resistance to biotic or a-biotic stress,
increased yield, increased yield quality, preferred growth pattern and
the like. According to additional embodiments, the encoded protein
products are useful in the cosmetic or pharmaceutical industry. According
to still further embodiments, the encoded protein enhances the production
of desired metabolites within the plant cells and tissues.

[0028] According to yet other embodiments the heterologous DNA dose not
encode a desired product but is present merely as a label to the origin
of the seed, or to the plant grown from said seed, for example to prevent
illegal distribution of proprietary seeds, plants and tissues.

[0029] The heterologous DNA may be transiently expressed in the cells and
cells derived therefrom or it may be incorporated into the cell genome.
The heterologous DNA may be present in the cytoplasm of the plant cell,
in its organelles or in the nucleolus as an integrated or non-integrated
DNA sequence.

[0030] According to certain currently preferred embodiments the DNA
construct is a Geminivirus-based construct designed such that the
heterologous DNA is not incorporated into the cell genome but the
construct is capable of replicating and spreading within the cells of the
plant grown from the seed comprising said heterologous DNA.

[0031] Any priming system and conditions as is known to a person skilled
in the art can be used according to the teachings of the present
invention, including systems comprising aqueous solutions with adequate
osmotic potential and systems comprising solid particulates. The water
potential of the medium enables water uptake that is insufficient for
complete seed imbibitions, and allows only the initial stages of
germination but not radicle emergence (completion of germination). The
system and conditions are typically selected according to the species of
the plant seed.

[0032] According to yet additional embodiments, the present invention
provides a seed produced by the methods of the invention, the seed
comprises a virus-based DNA construct comprising heterologous DNA, and
plants or parts thereof produced from said seed.

[0033] According to certain embodiments, the plant seed is of a monocot
origin. According to other embodiments, the plant seed is of a dicot
origin.

[0034] Other objects, features and advantages of the present invention
will become clear from the following description and drawings.

[0036] FIG. 2 is an illustration of the Geminivirus based constructs used
in the seed priming experiments described herein. FIG. 2A: IL-60-BS. FIG.
2B: pIR-GUS.

[0037] FIG. 3 shows the presence of GUS sequences in true tomato leaves
following seed priming in the presence of the Geminivirus based
constructs, detected by PCR. The dashed and solid arrows indicate the
position of a size marker of 1,500 bp and the position of GUS (ca. 1,800
bp) respectively (lane 1). Lane 8 and 12 are negative controls: PCR from
plants grown from non-treated seeds and from plant from seeds imbibed
with no DNA construct, respectively.

[0038] FIGS. 4A and 4B show cross sections of tomato leaves stained for
GUS activity following seed priming in the presence of IL-60-BS and
pIR-GUS (FIG. 4B mag. X40)

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0039] The term "priming" as used herein refers to the pre-sowing
restricted hydration treatment of seeds to improve germination and
seedling establishment and to the biochemical, physiological and
biological processes occurring within the seed prior to full hydration
and germination. The priming process controls and manipulates the water
availability to the seeds such that water availability suffice to
initiate and affect the early events of germination, but is not
sufficient to permit radicle emergence. Depending of the plant species
and growers requirements, the above-described controlled hydration is
subsequently followed by partial or complete drying, such that the seeds
can be further stored for short or long time periods until sowing.

[0041] The term "conditions enabling priming" refers to suitable
conditions of medium composition (e.g. osmotic PEG or other components
solutions, solid water absorbents, and more); duration; temperatures;
light/dark regimes; and aeration (e.g., stirring, agitation, bubbling,
etc.) to induce seed priming using any technique as is known in the art
for controlled water uptake.

[0042] The term "treated plants" refers to plants into which the
heterologous DNA has been introduced, regardless if it has been
integrated into the plant genome, into its organelles, or remained free
in the cytoplasm, or resided as an episome in the nucleus without
integration.

[0043] The term "plant" is used herein in its broadest sense. It includes,
but is not limited to, any species of woody, herbaceous, perennial or
annual plant. It also refers to a plurality of plant cells that are
largely differentiated into a structure that is present at any stage of a
plant's development. Such structures include, but are not limited to, a
root, stem, shoot, leaf, flower, petal, fruit, any storage organ (e.g.,
tuber, bulb, corm, false stem, leaves etc). The term "plant tissue"
includes differentiated and undifferentiated tissues of plants including
those present in roots, shoots, leaves, pollen, seeds and tumors, as well
as cells in culture (e.g., single cells, protoplasts, embryos, callus,
etc.). Plant tissue may be within the plant, in organ culture, tissue
culture, or cell culture. The term "plant part" as used herein refers to
a plant organ or a plant tissue.

[0044] The term "gene" refers to a nucleic acid (e.g., DNA or RNA)
sequence that comprises coding sequences necessary for the production of
RNA or a polypeptide. The term comprises natural as well as man tailored
(synthetic) genes. A polypeptide can be encoded by a full-length coding
sequence or by any part thereof. The term "parts thereof" when used in
reference to a gene refers to fragments of that gene ranging in size from
a few nucleotides to the entire gene sequence minus one nucleotide. Thus,
"a nucleic acid sequence comprising at least a part of a gene" may
comprise fragments of the gene or the entire gene.

[0045] The term "gene" also encompasses the coding regions of a structural
gene and includes sequences located adjacent to the coding region on both
the 5' and 3' ends for a distance of about 1 kb on either. The sequences
which are located 5' of the coding region and which are present on the
mRNA are referred to as 5' non-translated (or untranslated) sequences (5'
UTR). The sequences which are located 3' or downstream of the coding
region and which are present on the mRNA are referred to as 3'
non-translated (or untranslated) sequences (3' UTR).

[0046] The term "nucleic acid" as used herein refers to RNA or DNA that is
linear or branched, single or double stranded, or a hybrid thereof. The
term also encompasses RNA/DNA hybrids.

[0047] The terms "heterologous DNA" or "exogenous DNA" refer to a
polynucleotide that is not present in its natural environment (i.e., has
been altered by the hand of man). For example, a heterologous DNA
includes a polynucleotide from one species introduced into another
species. A heterologous DNA also includes a polynucleotide native to an
organism that has been altered in some way (e.g., mutated, added in
multiple copies, linked to a non-native promoter or enhancer sequence,
etc.). Heterologous DNA may comprise gene sequences of plants bacteria
and mammal origin. The gene sequences may comprise cDNA forms of a gene;
the cDNA sequences may be expressed in either a sense (to produce mRNA)
or anti-sense orientation (to produce an anti-sense RNA transcript that
is complementary to the mRNA transcript). Heterologous plant genes are
distinguished from endogenous plant genes in that the heterologous gene
sequences are typically joined to nucleotide sequences comprising
regulatory elements such as promoters that are not found naturally
associated with the gene for the protein encoded by the heterologous gene
or with plant gene sequences in the chromosome, or are associated with
portions of the chromosome not found in nature (e.g., genes expressed in
loci where the gene is not normally expressed).

[0048] The term "construct" is used herein in its broad sense, referring
to an artificially assembled or isolated nucleic acid molecule which
includes the heterologous DNA interest. In general a construct may
include the heterologous DNA, typically a gene of interest, a marker gene
which in some cases can also be the gene of interest and appropriate
regulatory sequences. It should be appreciated that the inclusion of
regulatory sequences in a construct is optional, for example, such
sequences may not be required in situations where the regulatory
sequences of a host cell are to be used. The term construct includes
vectors but should not be seen as being limited thereto.

[0049] The term "operably linked" refers to the association of nucleic
acid sequences on a single nucleic acid fragment so that the function of
one is regulated by the other. For example, a promoter is operably linked
with a coding sequence when it is capable of regulating the expression of
that coding sequence (i.e., that the coding sequence is under the
transcriptional control of the promoter). Coding sequences can be
operably linked to regulatory sequences in a sense or antisense
orientation.

[0050] The terms "promoter element," "promoter," or "promoter sequence" as
used herein, refer to a DNA sequence that is located at the 5' end (i.e.
precedes) the protein coding region of a DNA polymer. The location of
most promoters known in nature precedes the transcribed region. The
promoter functions as a switch, activating the expression of a gene. If
the gene is activated, it is said to be transcribed, or participating in
transcription. Transcription involves the synthesis of mRNA from the
gene. The promoter, therefore, serves as a transcriptional regulatory
element and also provides a site for initiation of transcription of the
gene into mRNA. Promoters may be derived in their entirety from a native
gene, or be composed of different elements derived from different
promoters found in nature, or even comprise synthetic DNA segments. It is
understood by those skilled in the art that different promoters may
direct the expression of a gene in different tissues or cell types, or at
different stages of development, or in response to different
environmental conditions. It is further recognized that since in most
cases the exact boundaries of regulatory sequences have not been
completely defined, DNA fragments of some variation may have identical
promoter activity. Promoters which cause a gene to be expressed in most
cell types at most times are commonly referred to as "constitutive
promoters". New promoters of various types useful in plant cells are
constantly being discovered; numerous examples may be found in Okamuro J
K and Goldberg R B (1989) Biochemistry of Plants 15:1-82.

[0051] As used herein, the term an "enhancer" refers to a DNA sequence
which can stimulate promoter activity, and may be an innate element of
the promoter or a heterologous element inserted to enhance the level or
tissue-specificity of a promoter.

[0052] The term "expression", as used herein, refers to the production of
a functional end-product e.g., an mRNA or a protein

Preferred Modes for Carrying Out the Invention

[0053] The present invention provides methods for introducing heterologous
DNA into plant cells or tissue. The methods of the invention enable a
simple, easy to use, inexpensive, widely accessible, and efficient way to
produce seeds and plants comprising exogenous DNA. According to the
methods of the invention the desired DNA is introduced into the seeds and
then plants are grown from the seeds, thus increasing considerably the
efficiency of DNA introduction and saving on the costly and sometimes
difficult plant regeneration process.

[0054] According to one aspect, the present invention provides a method
for introducing heterologous DNA into at least one cell of a plant seed
embryo comprising contacting the plant seed with a priming medium
containing a virus-based DNA construct comprising the heterologous DNA
under conditions enabling priming, thereby obtaining a seed embryo
comprising the heterologous DNA.

[0056] According to certain embodiments, the method further comprises
providing the suitable conditions for subsequent seed germination and
growth so as to obtain a plant or part thereof comprising the
heterologous DNA.

[0057] The term "comprising the heterologous DNA" when used in reference
to a plant or seed refers to a plant or seed that contains at least one
heterologous DNA in one or more of its cells. The term refers broadly to
a plant, a plant structure, a plant tissue, a plant seed or a plant cell
that contains at least one heterologous DNA in at least one of its cells.
This term includes the primary cell to which the DNA was introduced and
cultures and plants derived from that cell without regard to the number
of transfers. All progeny may not be precisely identical in DNA content,
due to deliberate or inadvertent mutations. Mutant progeny that have the
same functionality as screened for in the originally cell to which the
DNA was introduced are included in the definition of transformants.

[0058] Introduction of a heterologous DNA into a cell may be stable or
transient. The term "transient" refers to the introduction of one or more
exogenous polynucleotides into a cell in the absence of integration of
the exogenous polynucleotide into the host cell's genome. This type of
DNA introduction may be also referred to as "transient transformation".
The term "transient transformant" thus refers to a cell which has
transiently incorporated one or more exogenous polynucleotides.
Transiently transformed cells are typically referred to as
"non-transgenic" or "non-genetically modified (non-GMO)". In contrast,
stable DNA introduction is referred to as "stable transformation"
resulting in "stably transformed" cell or tissue and refers to the
introduction and integration of one or more exogenous polynucleotides
into the genome of a cell. The term "stable transformant" refers to a
cell which has stably integrated one or more exogenous polynucleotides
into the genomic or organellar DNA (chloroplast and/or mitochondria).
Plants or parts thereof comprising cell stably transformed with exogenous
DNA are typically referred to as "transgenic plants", "transgenic plant
cell" or, in the context of the present invention "transgenic seeds".

[0059] The virus-based DNA construct of the present invention is capable
of systemic, symptomless spread in the plant to which it was introduced.
As used herein, the term "systemic, symptomless spread" refers to the
ability of the virus-based vector to spread, for example, from the embryo
cell to the developing leaf cells, without inducing the characteristic
pathogenic symptoms of the virus. The DNA construct may be further
transmitted to the offspring of the plant comprising the heterologous
DNA. Without wishing to be bound by any specific theory or mechanism of
action, such transfer may result from the spreading of the virus-based
DNA construct into the plant reproductive cells.

[0060] According to certain currently preferred embodiment, the DNA
construct is based on the Geminivirus genetic components, as disclosed in
International (PCT) Application Publication No. WO2007/141790
incorporated herein in its entirety by reference. The construct comprises
the heterologous polynucleotide sequence flanked by a non-continuous
nucleic acid sequences encoding a Geminivirus replicase.

[0061] According to other embodiments, the construct further comprises a
polynucleotide sequence encoding a modified Geminivirus coat protein
(CP). According to typical embodiments, the modified Geminivirus coat
protein has the amino acid sequence set forth in SEQ ID NO:3. According
to additional embodiments, the modified Geminivirus coat protein encoding
polynucleotide comprises a mutation or deletion in nucleotides encoding
the N-terminal 100 amino acids.

[0062] According to further typical embodiments the expression construct
further comprises a polynucleotide sequence encoding a modified
Geminivirus V2 protein. According to typical embodiments, the modified
Geminivirus V2 protein has the amino acid sequence set forth in SEQ ID
NO:4.

[0063] According to additional typical embodiments the expression
construct further comprises a polynucleotide sequence encoding a modified
Geminivirus C4 protein. According to typical embodiments, the modified
Geminivirus C4 protein has the amino acid sequence set forth in SEQ ID
NO:5. According to these embodiments, the modified Geminivirus C4 protein
includes a mutation or deletion.

[0064] It is to be explicitly understood that other virus-based construct
carrying long heterologous polynucleotides and capable of replicating and
symptomless spread are also encompassed within the teachings of the
present invention. As used herein, the term "long heterologous
polynucleotides" refers to polynucleotides at least 1 kb long, typically
at least 5 kb long more typically 6 kb long.

[0065] According to certain typical embodiments, the DNA construct is
based on the tomato yellow leaf curl virus (TYLCV). Examples of such
constructs are IL-60-BS (having the nucleic acid sequence set forth in
SEQ ID NO:2) and pIR-GUS (having the nucleic acid sequence set forth in
SEQ ID NO:8) as described in FIG. 2.

[0066] The Geminivirus based construct used according to the teachings of
the present invention are introduced into the seed embryo cells, and are
not integrated into the genome of the cells. The plant grown from the
seed comprising the heterologous DNA and the plant grown therefrom are
thus referred to as non-genetically modified plant.

[0067] The agricultural use of genetically-modified plants is a matter of
public debate and in many countries is unacceptable by law or regulation.
The main considerations voiced against the use of transgenic plants are
the fear of inappropriate selection of a transgenic lineage (due to
masked deleterious positional effects), possible cross-fertilization with
weeds and other crops, further genome alterations due to recombination
(especially when copies of endogenous genes are added) and possible
transduction of the foreign sequences to plant and soil microorganisms.
Introduction of antibiotic-resistant genes to food and the environment is
also a major concern.

[0068] Bio-safety and environmental aspects can only be concluded upon
following actual, carefully controlled, field tests over time. Clearance
to conduct such experiments depends on evaluation based on hard
laboratory data. One advantage of the methods of the present invention
arises from the finding that the TYCLV based constructs appear to be
environmentally-friendly and ready for bio-safety-evaluation field tests.
Geminiviruses are not seed-transmissible (Kashina et al. 2003.
Phytoparasitica 31:188-199). The vector forms IL-60-BS and pIR are not
insect-transmissible even when the plants were colonized with a large
number of insect vectors. Thus, the TYCLV based constructs are highly
suitable for plant transformation.

[0069] The presence of the heterologous DNA in the cell of a treated seed,
whether expressed transiently or stably integrated, can be verified by
employing any suitable method as is known to a person skilled in the art.
Stable transformation of a cell may be detected by isolating genomic DNA
and employing Southern blot hybridization with nucleic acid sequences
which are capable of binding to one or more of the exogenous
polynucleotides or employing polymerase chain reaction with appropriate
primers to amplify exogenous polynucleotide sequences. Expression of the
transformed DNA can be detected by for example, enzyme-linked
immunosorbent assay (ELISA), which detects the presence of a polypeptide
encoded by one or more of the exogenous polynucleotides or by detecting
the activity of the protein encoded by the exogenous polynucleotide.

[0070] Alternatively, the exogenous DNA can comprise a marker. A marker
provides for the identification and/or selection of a cell, plant, and/or
seed expressing the marker. A marker can encode a product, which when
expressed at a sufficient level, confers resistance to a selective agent.
Such markers and their corresponding selective agents include, but are
not limited to, herbicide resistance genes and herbicides; antibiotic
resistance genes and antibiotics; and other chemical resistance genes
with their corresponding chemical agents. Bacterial drug resistance genes
include, but are not limited to, neomycin phosphotransferase II (nptII)
which confers resistance to kanamycin, paromycin, neomycin, and G418, and
hygromycin phosphotransferase (hph) which confers resistance to
hygromycin B. Resistance may also be conferred to herbicides from several
groups, including amino acid synthesis inhibitors, photosynthesis
inhibitors, lipid inhibitors, growth regulators, cell membrane
disrupters, pigment inhibitors, seedling growth inhibitors, including but
not limited to imidazolinones, sulfonylureas, triazolopyrimidines,
glyphosate, sethoxydim, fenoxaprop, glufosinate, phosphinothricin,
triazines, bromoxynil, and the like.

[0071] Additional type of marker is a marker the expression of which can
be detected by following a biochemical reaction preferably producing
color, upon providing of an appropriate substrate. Examples are the GUS
(beta-glucuronidase) reporter system, luciderin-luciferase,
green-fluorescent protein (GFP) system and the like.

[0072] According to certain embodiments, the DNA construct further
comprises a regulatory element including, but not limited to, a promoter,
an enhancer, and a termination signal.

[0074] The "3' non-coding sequences" refer to DNA sequences located
downstream of a coding sequence and include polyadenylation recognition
sequences and other sequences encoding regulatory signals capable of
affecting mRNA processing or gene expression. The polyadenylation signal
is usually characterized by affecting the addition of polyadenylic acid
tracts to the 3' end of the mRNA precursor. The use of different 3'
non-coding sequences is exemplified by Ingelbrecht I L. et al. (1989.
Plant Cell 1:671-680).

[0075] The method of the present invention can be used for introducing any
polynucleotide the presence and/or expression of which is of interest,
including giving the plant desired property(ies) such as (a) resistance
to biotic and a-biotic stress conditions, e.g., to insects, nematodes,
diseases caused by viral, bacterial fungal and other pathogens pests,
resistance to specific herbicide(s) (b) adaptation to hostile conditions
such as salt tolerance, drought tolerance, cold and heat tolerance; (c)
improved yield quality traits, including but not limited to pigmentation,
firmness, type and content of ingredients including sugars, volatiles,
oils, fatty acids and/or acids, size, growth rate, dwarf growth habit,
time of emergence, time of fruit appearance and ripening; nutritional or
commercial value; (d) improved production of edible yields, or of
secondary metabolites, for the biopharmaceutical and cosmetic industries
either in the form of a protein, secondary metabolites and (e) obtaining
a desired growth habit, including determinate, semideterminate and
indeterminate (e.g. for tomato), dwarf and normal (e.g. for corn) and
plant vigor. The introduced DNA can also confer gene silencing such that
the heterologous DNA is in the form of antisense, siRNA, dsRNA; or for
producing raw materials for industries, including but not limited to
fiber, wood, oils, resins, etc. as well as other plant traits governed by
genetic factors, and/or interaction by genes and environment.

[0077] The expressed polynucleotide sequence could be a metabolic enzyme
for use in the food-and-feed sector. Examples include phytases (GenBank
Ace. No.: A 19451) and cellulases.

[0078] The expressed polynucleotide sequence can confer resistance to
viruses, fungi, insects, nematodes and other pathogens and diseases, by
directly attacking the pathogen, turning on the host defenses or by
leading to an accumulation of certain metabolites or proteins. Examples
include glucosinolates (defense against herbivores), chitinases or
glucanases and other enzymes which destroy the cell wall of parasites,
ribosome-inactivating proteins (RIPS) and other proteins of the plant
resistance and stress reaction as are induced when plants are wounded or
attacked by microbes, or chemically, by, for example, salicylic acid,
jasmonic acid or ethylene, or lysozymes from non-plant sources such as,
for example, T4-lysozyme or lysozyme from a variety of mammals,
insecticidal proteins such as Bacillus thuringiensis endotoxin,
α-amylase inhibitor or protease inhibitors (cowpea trypsin
inhibitor), lectins such as wheatgerm agglutinin, siRNA, antisense RNA,
RNAses or ribozymes. Further examples are nucleic acids which encode the
Trichoderma harzianum chit42 endochitinase (GenBank Ace. No.: S78423) or
the N-hydroxylating, multi-functional cytochrome P-450 (CYP79) protein
from Sorghum bicolor (GenBank Ace. No.: U32624), or functional
equivalents thereof.

[0083] The expressed polynucleotide sequence can be also used for
obtaining an increased storability in cells which normally comprise fewer
storage proteins or storage lipids, with the purpose of increasing the
yield of these substances for example acetyl-CoA carboxylase. Preferred
polynucleotide sequences are those which encode the Medicago sativa
acetyl-CoA carboxylase (accase) (GenBank Ace. No.: L25042), or functional
equivalents thereof.

[0085] Further examples of polynucleotide sequence which can be expressed
in the transformed plants of the present invention are mentioned for
example in Dunwell J M. 2000. J Exp Bot. 51:487-96.

[0086] The heterologous DNA transformed into the plant cell according to
the teachings of the present invention can also be employed for the
reduction (suppression) of transcription and/or translation of target
genes. Thus, the DNA construct can comprises heterologous DNA the
expression of which brings about PTGS (post transcriptional gene
silencing) or TGS (transcriptional silencing) effects and thus a
reduction of the expression of endogenous genes. Such reduction can be
achieved for example by expression of an antisense RNA or of a
double-stranded RNA, each of which has homology with the endogenous
target gene to be suppressed. Also, the expression of a suitable sense
RNA can cause a reduction in the expression of endogenous genes, by means
of what is known as co-suppression (EP Application Publication No.
0465572). Particularly preferred is the expression of a double-stranded
small interfering RNA (siRNA) for reducing the gene expression of a
target gene via RNA interference (RNAi). Methods for inhibiting
individual target genes using RNA with double-stranded structure, where
the target gene and the region of the RNA duplex have at least partial
identity are known to a person skilled in the art.

[0087] The following provide examples for applications where reduction of
gene expression can be obtained by transforming plant seed with an
appropriate heterologous DNA according to teachings of the present
invention.

[0088] Delayed fruit maturation or a modified maturation phenotype
(prolonged maturation, later senescence) can be achieved for example by
reducing the gene expression of genes selected from the group consisting
of polygalacturonases, pectin esterases, β-1,4)glucanases
(cellulases), β-galactanases (β-galactosidases), or genes of
ethylene biosynthesis, such as 1-aminocyclopropane-1-carboxylate
synthase, adenosylmethionine hydrolase (SAMase),
aminocyclopropane-1-carb-oxylate deaminase,
aminocyclopropane-1-carboxylate oxidase, genes of carotenoid biosynthesis
such as, for example, genes of pre-phytoene biosynthesis or phytoene
biosynthesis, for example phytoene desaturases, and O-methyltransferases,
acyl carrier protein (ACP), elongation factor, auxin-induced gene,
cysteine(thiol) proteinases, starch phosphorylases, pyruvate
decarboxylases, chalcone reductases, protein kinases, auxin-related gene,
sucrose transporters, meristem pattern gene. Further advantageous genes
are described for example in International (PCT) Applications Publication
Nos. WO 91/16440, WO 91/05865, WO 91/16426, WO 92/17596, WO 93/07275 or
WO 92/04456. particularly preferred is the reduction of the expression of
polygalacturonase for the prevention of cell degradation and mushiness of
plants and fruits, for example tomatoes. Nucleic acid sequences such as
that of the tomato polygalacturonase gene (GenBank Acc. No.: x14074) or
its homologs can preferably used for this purpose.

[0089] The reduction of the gene expression of genes encoding storage
proteins has numerous advantages, such as, for example, the reduction of
the allergenic potential or modification regarding composition or
quantity of other metabolites, such as, for example, oil or starch
content.

[0090] Resistance to plant pathogens such as arachnids, fungi, insects,
nematodes, protozoans, viruses, bacteria and diseases can be achieved by
reducing the gene expression of genes which are essential for the growth,
survival, certain developmental stages (for example pupation) or the
multiplication of a specific pathogen. Such a reduction can bring about a
complete inhibition of the abovementioned steps, or else a delay of same.
They can take the form of plant genes which for example make possible the
penetration of the pathogen, but may also be homologous pathogen genes.
The transformed and expressed heterologous nucleic acid sequence (for
example the double-stranded RNA) is directed against genes of the
pathogen, such that the pathogen life cycle is interrupted.

[0091] Virus resistance can be achieved for example by reducing the
expression of a viral coat protein, a viral replicase, a viral protease
and the like. A large number of plant viruses and suitable target genes
are known to the skilled artisan.

[0095] A plant seed is a complete self-contained reproductive unit
generally consisting of a zygotic embryo resulting from sexual
fertilization or through asexual seed reproduction (apomixis), storage
reserves of nutrients in structures referred to as cotyledons, endosperm
or megagametophytes, and a protective seed coat encompassing the storage
reserves and embryo. In nature, maturation of plant seeds is usually
accompanied by gradual loss of water over a period of time to levels
between 5-35% moisture content. Once these low moisture levels are
achieved, plant seeds can be stored for extended periods.

[0096] Germination of sexual zygotic and apomictic plant seeds is
generally triggered by one or more environmental cues such as the
presence of water, oxygen, optimal temperature or cold/hot treatment, and
exposure to light and its duration. Seeds germinate by means of a series
of events which commence with the uptake of water (imbibition) by a
quiescent dry seed and then subsequently proceed through various
biophysical, biochemical and physiological events which ultimately result
in the elongation of the embryo along its axis and development of the
offspring.

[0097] The continuous process of seed germination may be divided into
three phases. Phase one is referred to as imbibition and is characterized
by a rapid initial intake of water into the seed. Other significant
events occurring in phase one are the initiation of repair of damage
nuclear and mitochondrial DNA, which may have occurred during seed
desiccation and/or the maturation process, and subsequent commencement of
protein synthesis facilitated by existing mRNA.

[0098] Phase two is characterized by a significant reduction in the rate
of water uptake (i.e., imbibition has been completed). This is
accompanied by activation or de novo synthesis of enzymes that specialize
in hydrolyzing the complex storage reserves of carbohydrates, proteins,
and lipids in the embryo and the cotyledons or megagametophytes. The
hydrolysis of these complex storage reserves provides the substrates
required for the respiration and growth of the seed embryos.

[0099] Phase three is characterized by a second rapid increase in the rate
of water uptake. Water absorbed during phase three is used primarily for
the initiation of meristematic cell division at the root and shoot apices
of the embryo, and for uptake into the cells along the embryonal axis.
Water taken up by the axial cells of the embryo applies turgor pressure
which results in axial cell elongation. The net effect is that the embryo
elongates to the point of emergence through the seed coat. Protrusion of
a shoot or root radicle through the seed coat signifies the completion of
germination and the onset of seedling growth and development.

[0100] The speed and success for germination of seeds varies considerably
depending on various factors such as the residual influence of
environmental conditions in which the seed developed and maturated, the
amount of storage reserve compounds synthesized during the seed
maturation process, the duration of storage, the quality of the storage
environment (e.g., temperature and humidity) and the environmental
conditions prevailing during the germination processes. From a commercial
perspective, it is desirable to reduce the risk of germination failure
and to ensure that seeds emerge and germinate rapidly and uniformly.

[0101] The commercial need for optimum seed germination performance has
led to the development of processes known in the art for zygotic seeds as
"seed priming". This term may be defined as the limited uptake of water
that is sufficient to initiate the early events of germination, but not
sufficient to permit radicle protrusion, preferably followed by drying.
Several principle techniques are used commercially to accomplish seed
priming. Regardless of the particular method used, the fundamental
principles of seed priming are that: (1) the preliminary stages of
germination are activated specifically and exclusively through
controlling the availability of water to the seeds, and (2) the
germination processes initiated through an external priming process are
subsequently arrested by a desiccation or partial desiccation step.

[0102] Unexpectedly, the present invention now shows that including a
virus-based DNA construct in the seed priming medium results in the
uptake of the DNA by the seed such that the DNA enters the seed cells.
Without wishing to be bound by any specific theory or mechanism of
action, this phenomenon may be attributed to viral elements enabling the
introduction of the heterologous DNA into the seeds cells.

[0103] Any method for seeds priming as is known to a person skilled in the
art can be used according to the teachings of the present invention.
Priming can be performed under a variety of temperatures and aeration
(e.g., stirring, agitation, bubbling, etc.) using any of the techniques
for controlled water uptake: priming with solutions (inorganic, e.g.,
salts/nutrients, or organic, e.g., PEG) or with solid particulate systems
or by controlled hydration with water as described, for example, in
Taylor, A G. et a11998. Seed Science Technology 8:245-256).

[0104] A priming matrix is characterized by its effective osmotic
potential. An effective osmotic potential typically lowers the water
potential available for seed imbibitions allowing or causing a limited
amount of water to move into the seed to a level sufficient for initial
steps of germination without actual protrusion of the radical, i.e., to
prime the seed. Seeds germination occurs only when water available to the
seed reaches a potential sufficient for physiological development, which
varies between plant species. Typically this value falls between 0 and -2
mPa. Many priming matrices that provide an appropriate osmotic potential
are being used, including water, water with one or more solutes, solid
matrices, and the like. For example, the priming matrix may comprise an
aerated solution of osmotic material, of organic nature such as
polyethylene glycol (PEG) (see U.S. Pat. No. 5,119,598), glycerol,
mannitol, or inorganic salt (or combination of salts) such as potassium
phosphate, potassium nitrate, and the like. Alternatively, seeds may be
primed using a solid matrix. A solid matrix material should have a high
water holding capacity to allow seeds to imbibe. In this method, the
priming matrix can comprise an absorbent medium such as clay,
vermiculite, perlite, saw dust, corn cobs, and/or peat to absorb water
and then transfer it to the seed (e.g., U.S. Pat. No. 4,912,874). The
extent of hydration is controlled by altering the water content of the
medium and the medium/seed ratio. Methods are also known to imbibe seeds
in a slurry of PEG 6000 and vermiculite, or other matrices (e.g., U.S.
Pat. No. 5,628,144). In still other methods, priming employs a
semi-permeable membrane that mediates the transfer of water from a
solution characterized by a given osmotic pressure to the seed (e.g.,
U.S. Pat. No. 5,873,197). In other methods, ultrasonic energy can be used
to assist in the priming process (e.g., U.S. Pat. No. 6,453,609).
Optionally a variety of additives, chemicals, and/or compounds can be
included in the priming matrix, including surfactants, selective agents,
fungicides, agents to modify osmotic potential, osmotic protectants,
agents to aid drying or protect the seed during drying, agents to enhance
seed processing, agents to extend storage shelf-life, agents to enhance
coating and/or perfusion, agents to enhance germination of the seed, and
the like. Fungicides can be included in the priming matrix, for example,
thiram, captan, metalaxyl, pentachloronitrobenzene, fenaminosulf,
bactericides or other preservatives. In addition, various growth
regulators or hormones, such as gibberellins or gibberellic acid,
cytokinins, inhibitors of abscissic acid, 2-(3,4-dichlorophenoxy)
triethylamine (DCPTA), potassium nitrate, and ethaphon can also be
present in the priming matrix. Other optional agents include glycerol,
polyethylene glycol, mannitol, DMSO, Triton X-100, Tween-20, NP-40, ionic
compounds, non-ionic compounds, surfactants, detergents, and the like. A
time sufficient to produce a primed seed allows pre-germinative metabolic
processes to take place within the seed up to any level including that
immediately preceding radicle-emergence. The time to produce a primed
seed is dependent on the specific seed variety, its state or condition,
and the water potential of the priming matrix. While typical water
amounts and media water potentials for given seed types are already
generally known for some seeds, it is frequently best to test a small
sample of a new seed over a readily determined range of osmotic
potentials and temperatures to determine what conditions of temperature,
water potential, and time provide appropriate imbibing of the seed and
resultant pre-germination events. The temperature at which the priming
methods are carried out may vary with the seeds to be treated, but
typically is between 18° C. to 30° C. The primed seeds may
be retained in the priming matrix through germination as denoted by
radical emergence. Seed produced by this method may be further dried
(e.g., as in U.S. Pat. No. 4,905,411).

[0106] After priming, the seeds may be allowed to germinate, or the primed
seeds can be dried. The appropriate conditions (temperature, relative
humidity, and time) for the drying process will vary depending on the
seed and can be determined empirically (see, for example, Jeller et al.
2003. ibid). Drying primed seed includes a superficial drying of the seed
or, alternatively, drying the seed back to its original water content.
The dried seeds can be immediately germinated or can be stored under
appropriate conditions. Germination conditions for various seeds are
known. One factor in determining appropriate germination conditions is
the threshold germination temperature range, which is the range of
temperatures for a species within which seeds of that species will
germinate at a predetermined moisture level and with adequate oxygen.
Another factor is the threshold germination moisture range, which is the
range of moisture for a species within which seeds of the species will
germinate at a given temperature and with adequate oxygen. Threshold
germination temperature range and/or threshold germination moisture range
values are known for various seeds, as are methods to empirically
determine these conditions for any given seed and variety.

[0107] The method of the invention greatly reduces the time required for
introduction of heterologous gene(s) into the plant cells, generation,
selection and propagation of the plants of interest. The potential for
somaclonal variation caused by tissue de-differentiation in culture is
nullified. When Geminivirus-based construct is used, the plants contain
foreign DNA, as every tissue infected with benign microorganisms does,
and yet the treated plants are not genomically modified. The method of
the invention can cut drastically the time and cost of breeding, since it
facilitates the expression of gene(s) of interest in any desired plant
variety, thus adding needed traits such as resistance/tolerance to
biotic/a-biotic stress conditions, improving yield and quality for any
given environment and market and the like, as long as genes controlling
the desired trait are known. Similarly, the heterologous DNA introduced
according to teachings of the present invention can result in silencing
of certain process(s) when so required (e.g., development of green back
in tomato fruit is desired in Italy but is unacceptable in the USA).

[0108] The following examples are presented in order to more fully
illustrate some embodiments of the invention. They should, in no way be
construed, however, as limiting the broad scope of the invention. One
skilled in the art can readily devise many variations and modifications
of the principles disclosed herein without departing from the scope of
the invention.

[0110] Batches of 50 tomato seeds were imbibed in a solution composed of
0.1M Ca(NO3)2; 0.1M MES (4-morpholineetahnsulfonic acid); 15 mM
MgCl2 and 25% (solution T2) or 20% (solution T3) of PEG 8000. The
osmotic pressure of solutions T2 and T3 was -1.25 mPa and -1.2 mPa
respectively. Untreated dry seeds of the same cultivar, same seed lot
were used as control. Treatment was carried out under constant
temperature of 20° C. with a 12 hour light regimen daily, and
stirred gently for 4 or 6 days to obtain proper aeration. Seeds were then
placed on 3M filter paper supplemented with 3 ml sterile water and
germination rates were recorded daily. The data demonstrate (FIG. 1) a
significant increase in germination uniformity of primed seed in
comparison with control (successful priming).

Example 2

Introducing IL-60 Constructs into Seeds by Using Priming Medium

[0111] Tomato seeds were primed with a priming solution containing 25% PEG
and germinated as described in Example 1 above in the presence of 20, 40
and 60 μg of each of the IL-60-BS (SEQ ID NO:2) and pIR-GUS (SEQ ID
NO:8) DNA constructs. The constructs are illustrated in FIGS. 2A and 2B,
respectively. Constructs are also described in International (PCT) Patent
Application Publication No. WO 2007/141790, incorporated herein by
reference. Untreated dry seeds and seeds primed at the same conditions
but without the DNA construct served as control.

[0112] Six days after germination the seedlings were transplanted into
pots containing 10 liters soil and placed in a greenhouse at 25°
C. Twenty one days after planting, DNA was extracted from true leaves
(the third true leaf or above) and subjected to PCR with the following
primers for GUS assay:

[0113] Forward primer: ATTGATCAGCGTTGGTGGGA (SEQ ID NO:6) and reverse
primer: TGCGGTCGCGAGTGAAGATC (SEQ ID NO:7) designed to amplify the entire
gus gene present within the pIR-GUS DNA construct. Positive PCR reaction
confirmed the presence of a GUS DNA sequence in the true leaves. These
results show that the DNA constructs of the IL-60 based vectors were
introduced to the seed cell(s) using the above priming procedure. The
introduced DNA replicated, spread and was expressed throughout the plant
system. An example of the results of such assay is shown in FIG. 3.

Example 3

Expression of the Foreign Genes

[0114] The DNA constructs of the IL-60 family as detailed in Example 2
above (IL-60-BS in combination with pIR-GUS) facilitated the expression
of the foreign gus gene present within the pIR-GUS DNA construct and
introduced via priming.

[0115] Tomato leaves from the plant obtained as described in Example 2
above were stained for beta-glucuronidase (GUS) according to Jefferson R
A. et al. (1987. EMBO J 6: 3901-3907).

[0117] Priming was conducted as in example 2 with the basic priming
solution containing 5% PEG and the addition of 75% dimethylsulfoxide
(DMSO) as to obtain final osmotic potential of -0.131 mPa (1.296 Atm.,
according to Christopher et. al Macromolecules 2003. 36:6888-6893). In
this modified priming solution, the IR-GUS DNA was introduced and
replicated as indicated by GUS expression as described in Examples 2 and
3 above.

[0118] The foregoing description of the specific embodiments will so fully
reveal the general nature of the invention that others can, by applying
current knowledge, readily modify and/or adapt for various applications
such specific embodiments without undue experimentation and without
departing from the generic concept, and, therefore, such adaptations and
modifications should and are intended to be comprehended within the
meaning and range of equivalents of the disclosed embodiments. It is to
be understood that the phraseology or terminology employed herein is for
the purpose of description and not of limitation. The means, materials,
and steps for carrying out various disclosed functions may take a variety
of alternative forms without departing from the invention.